Complementary DNA (cDNA) clones specific for phospholamban of sarcoplasmic reticulum membranes have been isolated from a canine cardiac cDNA library. The amino acid sequence deduced from the cDNA sequence indicates that phospholamban consists of 52 amino acid residues ,and lacks an amino-terminal signal sequence. The protein has an. inferred mol wt 6,080 that is in agreement with its apparent monomeric mol wt 6,000, estimated previously by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phospholamban contains two distinct domains, a hydrophilic region at the amino terminus (domain I) and a hydrophobic region at the carboxy terminus (domain II). We propose that domain I is localized at the cytoplasmic surface and offers phosphorylatable sites whereas domain II is anchored into the sarcoplasmic reticulum membrane.

Estrogens attenuate renal injury induced by ischemia/reperfusion (I/R), an effect that is related to nitric oxide production in the post-ischemic kidney. The compound 17beta-estradiol (E(2)-beta) acting via estrogen receptors (ERs) is known to activate endothelial nitric oxide synthase (eNOS) through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. We determined if this pathway contributes to the renoprotective effect of E(2)-beta in the uninephrectomized ischemia reperfusion rat model of acute renal injury. Treatment with E(2)-beta suppressed the I/R-induced increases in blood urea nitrogen, plasma creatinine, urine flow, and fractional excretion of sodium while augmenting creatinine clearance, renal blood flow, and urine osmolality, indicating attenuation of renal injury. Phosphorylation of Akt and eNOS protein was significantly increased 30-60 min after reperfusion in estradiol-treated compared to vehicle-treated rats. The protective effects of E(2)-beta and protein phosphorylation were reversed by the PI3K inhibitor wortmannin or the ER antagonist tamoxifen. Furthermore, the E(2)-beta-induced renoprotective effects were not seen in eNOS knockout mice with renal injury. We conclude that the E(2)-beta-induced renoprotective effect is due to activation of the PI3K/Akt pathway followed by increased eNOS phosphorylation in the post-ischemic kidney.

A gene coding for human growth hormone, which consists of 192 amino acids, was chemically synthesized.The synthesis entailed ligating 78 deoxyribooligonucleotides, which had been synthesized on polymer supports by the phosphotriester method with frequently occurring amino acid codons of Escherichia coli. The chemically synthesized gene was inserted into an E. coli plasmid downstream from the E. coli thp promoter, with a modified ribosome-binding region carried on pBR322. E. coli cells transformed with this recombinant plasmid synthesized 2.9 x 106 molecules per cell of human growth hormone upon induction. The induced polypeptide was identical with natural human growth hormone in size and in immunological properties, as well as in biological activity as examined by the tibial test with hypophysectomized rats.The improvement of techniques in chemical synthesis of deoxyribooligonucleotides has made possible the total synthesis of genes of 100-500 nucleotides (1-9). By this technique, genes with designed amino acid sequences can in principle be synthesized, and the products of their expression in bacteria can be examined. In the present work, a gene for human growth hormone (hGH) (10), which contains 191 amino acids and methionine, was totally synthesized by chemical means, using amino acid codons most frequently appearing in Escherichia coli (11-13) (Fig. 1). To establish a general procedure for efficient expression of genes for relatively large peptides, hGH was thought to be a suitable target. Use of the synthetic gene will provide sufficient sites for restriction enzymes, which should be useful in mutations of genes for replacement of protein domains. Rapid synthesis of 78 gene fragments of 7-26 bases was carried out, followed by sequential ligation to construct the gene of 584 base pairs (bp). This is the longest gene so far synthesized chemically. Separately, a modified trp promoter was constructed that allows expression of any synthetic genes with an initiation codon placed downstream of it. A recombinant plasmid carrying the promoter and the hGH gene produced the hormone in E. coli at a high level. The induced polypeptide was indistinguishable from the natural hGH in several features. The purified hormone was as active as the natural growth hormone in biological tests (14). MATERIALS AND METHODSDeoxyoligonucleotides. Deoxyoligonucleotides with a chain length of 7-26 bases were synthesized by the phosphotriester solid-phase method, using 1% cross-linked polystyrene (15) as the support. Altogether 78 chains were prepared by addition of dinucleotide blocks to the nucleosides linked through a 3'-succinyl group to the resin. Tetranucleotide blocks prepared by condensation of dimers in the liquid phase were used for the synthesis of the 25-mer (A-UO; Fig. 1). Sixteen dinucleotide blocks were prepared with mesitylenesulfonyl-3-nitro-triazolide (16) by methods essentially as reported (17) and purified mostly by reversed-phase chromatography (18) under conditions described (19,20). A typical synthesis of a 1...

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The mechanism of interaction of the operator DNA with the lambda‐cro repressor protein was investigated using proton n.m.r. and photo CIDNP. Three kinds of DNA duplexes, the lambda‐OR3 17‐mer, phi80‐OR2 19‐mer and CRP binding site 22‐mer, were prepared, and all of their imino proton resonances of the complexes with lambda‐cro were assigned to individual base pairs. By monitoring the assigned signals of the DNA fragments and lambda‐cro, it was found that in the complex of lambda‐cro with lambda‐OR3, two subunits of the cro dimer bind to the right and left halves of the OR3, respectively, and the bidentate binding induces a structural distortion in the middle of the 17‐mer. lambda‐cro itself also undergoes a conformational change including loosening of the dimeric form. In the complex of lambda‐cro with phi 80‐OR2, which has a 6‐bp sequence common to that of lambda‐OR3, one subunit of the cro dimer seems to bind specifically to the common part. However, there is only a slight conformational change in the cro dimer. In the mixture of the CRP binding site 22‐mer and lambda‐cro, soft contact without any conformational change was observed between them.